The present invention relates, in general, to lipid A 4xe2x80x2 kinase and, in particular, to a nucleic acid encoding lipid A 4xe2x80x2 kinase and to a method of producing lipid A 4xe2x80x2 kinase recombinantly using same. The invention further relates to methods of producing 4xe2x80x2 phosphorylated lipid A analogs using the recombinantly produced lipid A 4xe2x80x2 kinase.
Lipopolysaccharide (LPS) is the major glycolipid of the outer membrane of gram-negative bacteria. Lipid A, or endotoxin, is the hydrophobic anchor of LPS, and it is a potent immunostimulant. It appears to be responsible for many of the features of septic shock that can accompany severe gram-negative infections. Lipid A is a disaccharide of glucosamine that is phosphorylated at the 1 and 4xe2x80x2 positions and is acylated with R-3 hydroxymyristate at the 2, 3, 2xe2x80x2, and 3xe2x80x2 positions. In E. coli, two additional fatty acyl chains are also esterified to the 2xe2x80x2 and 3xe2x80x2 R-3 hydroxymyristate groups to form acyloxyacyl units.
Lipid A biosynthesis begins with the acyl-ACP dependent acylation of UDP-N-acetylglucosamine (Anderson et al, J. Biol. Chem. 260:15536 (1985), Anderson et al, J. Biol. Chem. 262:5159 (1987), Anderson et al, J. Biol. Chem. 268:19858 (1993), Williamson et al, J. Bacteriol. 173:3591 (1991), Raetz et al, Science 270:997 (1995)). Nine enzymes are required for the complete synthesis of Kdo2-lipid A (Raetz, J. Bacteriol. 175:5745 (1993), Raetz, Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996), Raetz et al, J. Biol. Chem. 265:1235 (1990)). Seven of the nine structural genes coding for the enzymes of lipid A biosynthesis in E. coli have been identified, however, the lipid A 4xe2x80x2 kinase gene has remained elusive (Raetz, Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996)). The 4xe2x80x2 kinase catalyzes the sixth step of the pathway (FIG. 2) (Ray et al, J. Biol. Chem. 262:1122 (1987)). It phosphorylates the 4xe2x80x2 position of a tetraacyldisaccharide-1-phosphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1, 4xe2x80x2 bis-phosphate, also known as lipid IVA (FIG. 2) (Ray et al, J. Biol. Chem. 262:1122 (1987), Raetz et al, J. Biol. Chem. 260:16080 (1985), Strain et al, J. Biol. Chem. 260:16089 (1985)).
Identification of the 4xe2x80x2 kinase gene has been hampered because mutants lacking the 4xe2x80x2 kinase have not been identified (Raetz, Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996)). Attempts to purify the kinase to homogeneity have been thwarted by the protein""s association with membranes and its instability in the presence of detergents (Ray et al, J. Biol. Chem. 262:1122 (1987), Hampton et al, Methods in Enzymology 209:466 (1992)).
The lipid A 4xe2x80x2 kinase can be used to make 4xe2x80x2-32P labeled lipid A precursors, such as [4xe2x80x2-32P]-lipid IVA and Kdo2-[4xe2x80x2-32P]-lipid IVA, for biochemical analyses of late pathway reactions. The 4xe2x80x2 kinase activity found in wild type E. coli membranes, however, is relatively inefficient and unstable, especially in the presence of low chemical concentrations of ATP. The inability to achieve high levels of 32P transfer makes it virtually impossible to use the 4xe2x80x2 kinase for phosphorylating DS-1-P analogs that are utilized less rapidly. Identification and overexpression of the 4xe2x80x2 kinase gene would facilitate the synthesis of 4xe2x80x2 phosphorylated lipid A analogs with activity as endotoxin antagonists or mimetics (Raetz, J. Bacteriol. 175:5745 (1993), Raetz, Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996)). The present invention provides a nucleic acid encoding lipid A 4xe2x80x2 kinase and a method of producing 4xe2x80x2 kinase using same.
It is a general object of the invention to provide a nucleic acid encoding lipid A 4xe2x80x2 kinase.
It is another object of the invention to provide a method of producing lipid A 4xe2x80x2 kinase recombinantly.
It is a further object of the invention to provide a recombinantly produced lipid A 4xe2x80x2 kinase.
It is a further object of the invention to provide a method of producing lipid A analogs suitable for use as endotoxin mimetics or endotoxin antagonists, using lipid A 4xe2x80x2 kinase.
The foregoing objects are met by the present invention which relates to a nucleic acid encoding 4xe2x80x2 kinase, to an expression construct comprising that nucleic acid and to a host cell into which the construct has been introduced. The invention further relates to a method of producing 4xe2x80x2 kinase using such host cells and to the 4xe2x80x2 kinase produced thereby. In addition, the invention relates to the production of 4xe2x80x2 phosphorylated lipid A analogs using the recombinantly produced 4xe2x80x2 kinase.
Further objects and advantages of the invention will be clear from the description that follows.